We have been interested in the protein CTCF, which we first identified some years ago as having properties of an insulator, blocking interaction between enhancers and promoters when placed between them. We demonstrated that this activity plays an important role in regulating parent of origin allele-specific gene expression at the Igf2/H19 imprinted locus. Work in other laboratories in recent years has shown that a principal mode of action of CTCF is to recruit cohesin which in turn stabilizes interactions between CTCF binding sites on DNA, leading to formation of loop domains. Depending on the geometry of the interactions such loops can either exclude an enhancer leading to insulation, or bring enhancer and promoter closer together, leading to activation. DNA within the cell nucleus is packaged into chromatin, and further organized into topologically associated domains (TADs) separating active and inactive genomic regions. The establishment and maintenance of TADs requires the protein CTCF, and we are interested in identifying and studying the interactions of CTCF with the protein and nucleic acid partners recruited for insulator function. We have shown that the N- and C-terminal domains that flank the DNA binding 11 zinc fingers of CTCF appear to be intrinsically disordered explaining, in part, the large number of CTCF binding partners identified in other studies. Current work focuses on further characterizing the physical nature of these domains, identifying partners that bind with high affinity, and studying the complexes formed. In one recent study we have identified and characterized CTCF sites within the HIV genome. The location of these sites presents unusual organizational problems. Our work with CTCF has led to an interest in the more general question of large scale genome structure within the nucleus. We have identified other proteins that appear to contribute to chromatin organization and to collaborate with CTCF. In other work we have continued to study genome organization in pancreatic beta cells, with emphasis on interactions between the insulin gene locus and sites on other chromosomes that contain genes which are involved in insulin secretion. We have participated in a collaboration investigating a possible role for SNPs affecting CTCF binding strength in expression of a gene associated with major mental illnesses. We have also begun a study of the organization of non-ribosomal genes within the nucleolus. All of these results relate to the role of chromatin structure, histone modifications, and long range organization of the genome in cell function, and are in turn related to questions of normal and abnormal cell metabolism and cell division.